By utilization of heterosis of rice, China succeeded in developing of hybrid rice and this made the rice production increased substantially in total. Currently, China is large scaly exploring two-line system hybrid rice after success in utilization of the three-line heterosis based on nucleo-cytoplasmic interaction male sterility. The hybrid seeds of the two-line system are produced by photo- and thermo-sensitive genic male sterile line. But the sterility of this male sterile line is easily affected by environmental temperature, especially the midsummer low temperature that may cause its fertility restored. This thus results in a potential risk: when seed production of two-line system meets low temperature, the harvest seeds may comprise of false hybrid seeds (selfing seeds from male sterile line) mixed in the real hybrid seeds. Once presence of this mixture of the hybrid seeds with selfings from female parent and fail to eliminate them, it will causes a great loss to seed or field production. Guangxi in 1989 and Hunan in 1999 suffered such big loss right because of these.
The existing data proved that the seed purity declined 1 percent and yield of hybrid rice would reduce 75 kg per hectare. This is why the seed standard published by Chinese Ministry of Agriculture stipulates that the purity of hybrid seeds has to stand over 98%. Not only the photo- and thermo-sensitive male sterile line produced the two-line hybrid seeds have the selfing contamination problems, but so do the hybrid seeds when produced by incomplete male sterile lines, such as their sterility governed by nucleic major gene/s that have modifier gene/s participated in, or resulted from artificial chemical emasculation, or from environment sensitive nucleo-cytoplasmic interaction, or from artificially developed aneuploid. Many attempts have been made to eliminate such selfing mixture problems and thus to have the crop heterosis utilization establish on a more reliable foundation. However, in view of the characteristic of herbicide tolerant/resistant being widely used in the modern crop breeding program, not a few of scientists also attempt to utilize the wild-type rice that have the resistance to both bentazon and sulfonylurea to solve the above mentioned selfing mixture problems during the hybrid seed production.
There are two main categories of selective herbicides applied to rice. The former one is a benzothiadiazinone contact herbicide, such as bentazon, and its effective component can be absorbed through roots and leaves of crop. It kills the overwhelming dicot plants and sedges in most gramineous species excluding leguminous while it is harmless to rice. The herbicide mechanism of this herbicide is to inhibit Hill reaction in Photosynthesis. But the endogenous gene that is resistant to such herbicide of bentazon has not yet been cloned from plants so far. The later one is sulfonylurea-like herbicides explored by DuPont Company, which represent a new category of super effective herbicides characterized in high selectivity, broad spectrum, low poisons, and interior absorption. Among which, the tribenuron-ethyl, and bensulfuron-methyl and their complex formula are the most widely used herbicides in paddy field in China at present. The most notable characteristic of the sulfonylurea herbicides is the high activity, which makes their on-use dosage usually within 5-100 gram per hectare. The sulfonylurea-like herbicides are the acetolactate synthase (ALS) inhibitors, which have special effect to many annul or perennial weeds, especially the broadleaf weeds and are already widely used to eliminate the weeds growing in the field of rice, wheat, soybean, corn, canola, and lawn and other non-cultivated land. The DuPont Company has explored several sulfonylurea-resistant genes. One of these genes is SURB-Hra cloned from a tobacco ALS mutant. The SURB-Hra expresses resistance is because the mutated ALS is insensitive to the sulfonylurea. This gene has been applied to various crops including cotton and soybean (U.S. Pat. Nos. 5,013,659, 5,084,086, 5,141,870, 5,378,824, 5,605,011); Another sulfonylurea resistant gene developed by DuPont Company is the P450 su1 gene isolated from soil bacteria. The mode of action of this gene is to accelerate the metabolism of sulfonylurea to non-toxic. DuPont has made great efforts on the studies of this P450 gene and its application (see patent U.S. Pat. No. 5,349,127 for relevant information). In a patent (WO9708327) document, Japanese Nissan Chemical Corporation also publicized an ALS gene that was isolated from the cDNA of Kochia coparia, a kind of dicotyledonous broadleaf plant, having the function to make the transgenic plant resistant to sulfonylurea.
At present, two major approaches are applied to develop herbicide tolerant or resistant crops: the first one is by use of traditional physical/chemical mutagenesis to obtain the crop mutant capable to resistant or tolerant to herbicides; the second one is through recombinant DNA technology to introduce herbicide tolerant or resistant gene/s into the existing species to create the new materials tolerant or resistant to herbicides. Among which, the latter approach is the most widely used method. Currently, to enhance the crop herbicide tolerance or resistant, there are also two strategies involving in use of this recombinant DNA technology: first is to modify the herbicide target protein and make it insensitive to herbicide or over expressed to let plant still capable to normally metabolize the herbicide after absorption; second is to introduce in an new enzyme or enzyme system, such as P450 monooxygenase (Wang Guanlin and Fang Hongjun, 1998), to degrade or detoxify the absorbed herbicide before it functions.
Wild-type rice is naturally resistant to bentazon and sulfonylurea herbicides. Mori, a Japanese scholar and Zhang Jiwen et al from Hubei Academy of Agricultural Sciences made two recessive bentazon-sensitive-lethal mutants Norin 8m (Mori, 1984) and 8077S (Zhang Jiwen and Wu Xiaozhi, 1999) using X-ray radiation to treat Norin 8 and W6154, respectively. Based on these, Zhang et al (2001) further developed a selfing seed removal technology system to ensure the hybrid seed purity by use of the recessive mutant locus to tag the thermo-sensitive male sterile line. Since such a germplasm source plays an important role in the seed purity security and ensuring system of the two-line hybrid rice, e.g. use of on-8077S mutant locus tagged two-line's male sterile line could largely reduces the risk of seed production of the two-line hybrid rice, it is thus highly recognized by rice breeders and seed enterprises. However, since this hybrid seed purity-ensuring system functions only after seed-harvesting, even though its effect is not bad, it is hardly approved by seed administration department because what the technology ensured is the non-purified seeds and these are not according with the government-published seed purity standard before sale. Therefore, there is a need for creating a new mechanism to remove mixtures and to ensure the purity. However, the problem is that the gene controlling this trait has not yet been cloned for many years. It thus makes people have no way to conduct manipulation and further utilization of this trait, which has already become a major technical obstacle in this field.
Based on above situations, the present inventor used two existing bentazon sensitive lethal mutants as materials to conduct fine mapping of their mutant loci and finally cloned their common wild-type alleles through deepgoing investigation. Further on the base of this, the inventors developed several useful methods and techniques. These include a method for development of chemically supplemented emasculation and thermo-sensitive male sterile line, a genetic manipulation technique of double- or multi-sites targeted co-modification, a new approach for investigating biological function of plant genes, and a new technology for genetic improvement of plant traits. Therefore, this invention not only solves the selfing mixture problems during the hybrid seed production, but also provides the useful means with broad application perspective for investigating biological functions of genes and improving the biological traits genetically.